Investigating the function of the autism spectrum disorder risk genes RFX3 and RFX4 during human cortical development

Pathogenic mutations in the genes encoding Regulatory Factor X3 and X4 (RFX3/RFX4) transcription factors (TFs) were recently identified contributors to autism spectrum disorder and other neurodevelopmental disorders (NDDs). In preliminary work, I identified RFX4 as a TF that binds to cis-regulatory elements to control human cortical interneuron development, defining roles for RFX3 and RFX4 in regulating interneuron progenitor specification and differentiation. While prior work had demonstrated a role for RFX4 in ciliogenesis, its role in human brain development and contribution to NDDs remains undefined. Therefore, my proposed work will define the role that RFX4 plays in human cortical development to determine how pathogenic RFX4 mutations affect these processes. Toward that end, I derived human pluripotent stem cell (hPSC) models carrying pathogenic RFX4 heterozygous and homozygous loss of function (LOF) and patient-specific mutations. Using these models, I have demonstrated that RFX4 LOF causes dosage-dependent spontaneous differentiation of neural progenitors; likely due in part to a loss of RFX4-mediated repression of pro-neuronal genes. Furthermore, I have identified a regulatory relationship between RFX3 and RFX4, with RFX4 LOF resulting in loss many RFX3 binding sites, particularly at genes involved in synapse development. From these and other preliminary data, I hypothesize that RFX3 and RFX4 restrain neuronal differentiation in progenitors, with loss of either TF causing precocious neuronal differentiation resulting in altered maturation and function, likely major contributors to the NDD phenotypes found in human patients. In Aim 1, I will examine how RFX4 pathogenic mutations contribute to NDD etiology, defining temporal requirements for RFX4 and identifying consequences of RFX4 pathogenic mutation through a combination of cellular phenotyping and transcriptomic analyses across 2- and 3-diminsional models. I will also determine how pathogenic mutation affects RFX4 genome-wide occupancy and gene regulation by integrating RFX4 binding and transcriptomic data. Finally, I will define how RFX4 LOF mutation impacts neuronal maturation and neuronal network formation, providing a foundation for understanding the etiology of NDD pathogenesis resulting from RFX4 mutation which can be therapeutically targeted. In Aim 2, I will characterize the transcriptional interplay between RFX3 and RFX4, which is suggested from my preliminary data, examining the impact of RFX3 LOF upon RFX4 binding. This work will elucidate the basis of the proposed synergistic relationship between RFX3 and RFX4 in neuronal gene repression and will determine how transcriptional interplay between RFX3 and RFX4 regulates neuronal differentiation, maturation, and function, exploring convergent RFX3 and RFX4 LOF phenotypes that may contribute to NDD etiology. Together, my work under this proposal will provide novel insights into the etiology of RFX-associated NDDs and provides well characterized cellular phenotypes that can be used as a platform to develop gene targeted and pharmacological interventions to treat RFX mutation-associated NDDs. . Project Number: 1F31MH141956-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Mental Health (NIMH) | Principal Investigator: Julianna Determan | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $36,673 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F03A-E (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11241478